Single CRP antigen detection with TiN plasmonic biosensor
C-reactive protein (CRP) [1] is a typical indicator of body tissue inflammation. It is made by the liver and its concentration increases with IL6 secretion by macrophages white blood cell and T cells. The role of CRP is to bind to to lysophosphatidylcholine (LPC) [2] expressed on the surface of dead or dying cells. Since LPC acts as a find-me signal, and LPC is released by dead cells to recruit phagocytes. Our body immune system instructs macrophages white blood cells to phagocytose the dead cells. The CRP molecular weight is about 115KDa. To test the analytical performance of our QPLoC system, the CRP antibody is functionalized onto our TiN plasmonic biochip. Whereas the CRP antigen is repeatedly diluted to known concentrations for detection. The ultimate limit of detection is 3.26 aM (attomolar i.e. 3.26E-18 mole per liter or about 2,000 copies per milliliter). Since our liquid sample volume is 0.36 milliliter which is about 720 CRP antigen molecules in total or 5 CRP molecules in every microwell. This is a significant improvement over existing SPR systems which usually attains picomolar i.e. 1E-12 mole per liter. Our QPLoC system outperforms existing systems by six orders of magnitude, and the attomolar sensitivity is repeatable. Our QPLoC sensing protocol remains label-free with fairly straightforward sample preparation. The CRP antigen initial concentration is 3.75 mg/mL and the CRP antibody initial concentration is 6.23 mg/mL.
These are the experimental steps:
- CRP antibody solution is diluted to 0.623 ug/ml with Phosphate-buffered saline (PBS) in EP tubes.
- Take 100ul of the antibody dilution solution from each of the antibody EP tubes and add it to the TiN biochip and slide the pipette on the chip surface to spread the antibody evenly on the chip surface. Incubate at room temperature for 30 minutes.
- Shake off the antibody reagents on the chip surface, rinse 3-5 times with the prepared PB buffer, and finally spin dry.
- Add 1% Bovine Serum Albumin (BSA) as blocking agent to block the TiN biochip with the same steps as 2 and 3 above.
- Shake off the BSA solution, rinse 3-5 times with PBS buffer, dry in room temperature for 5 minutes.
- Use immediately or store in an aluminum foil bag in the refrigerator at 2-8°C.
- CRP antigen solution is repeatedly diluted in series i.e. 1/1E03 → 1/1E04 → 1/1E05 → 1/1E06→ 1/1E07 →1/1E08 → 1/1E09 → 1/1E10 → 1/1E11 → 1/1E12 → 1/1E13 → 1/1E14. With 1/1E13 dilution, the molar concentration is 3.26 attomolar or 1963 CRP antigen molecule per mL.
- Power up the QPLoC system and start the data acquisition software. After starting up, install the CRP antibody functionalized chip into the instrument fixture, place purified water on the sample position of the instrument, and start the degassing procedure to purge the biochip with purified water twice.
- Place the diluted antigen solutions on the sample position of the instrument starting with the lowest concentration, make sure that the sample inlet tube is underneath the sample liquid level then starts the data acquisition program which conduct the experiment automatically.
- The system records the first 100 frames to establish the baseline, then captures 600 frames for CRP antibody-antigen binding, finally another 100 frames are taken after flushing with purified water to confirm the success of binding. Total of 800 frames were recorded for each CRP antigen concentration.
- The system captures the phase image of the TiN biochip on CRP antigen-antibody binding at rate of 1 frame per second. The image size is 2,200 pixels square with total of 4.84 million pixels. For each microwell, it occupies about 8,000 pixels. The physical size of each pixel covers approximately 5 microns square.
- The phase map of every microwell is processed by a remote server and the history plot of each pixel is also extracted by our analysis software.
Results:
The following results show the attomolar sensitivity of our QPLoC biosensor. Figure 1 is the 2D map of phase change of a particular microwell after CRP antibody-antigen binding. Figure 2 is the 2D map of the same microwell after rinsing with purified water as reference. Figure 3 shows the history plot of a particular pixel from the 2D map with blue circles representing the raw data and the red curve is the B-splined fitted plot of the raw data set. Figure 4 is the normal distributions of the 2D phase maps of the same microwell after repeated experiments of 3.26 aM CRP antigen concentrations. Data sets fit 1, fit 2 and fit 4 of Figure 4 show similar trend and fit 3 is with purified water as reference. Significant change in the mean and variance of the distribution can be observed in Figure 4 with binding of just a few CRP antigen molecules. Thus, we claims that our QPLoC biosensor is capable of single CRP molecule detection in a microwell.
Figure 1. 2D phase map with 3.26 aM CRP antigen
Figure 2. 2D phase map with purified water
Figure 3. Pixel history plot of 800 frames with raw data and B-splined curve
Figure 4. Normal distribution fit of the 2D phase map from repeated CRP experiments and reference
References
[1] C-reactive protein, https://en.wikipedia.org/wiki/C-reactive_protein
[2] Lysophosphatidylcholine, https://en.wikipedia.org/wiki/Lysophosphatidylcholine